The proper regulation of transcription is essential to ensure that cells respond appropriately to intracellular cues and environmental variations. Temporal or spatial defects in gene expression directly impact cell growth and division, responses to stress and energy sources, and communication between neighboring cells. In higher eukaryotes, transcription plays a vital role in controlling differentiation into specific cell types, a process that can lead to uncontrolled cell growth and tumor formation when misdirected. A crucial part of understanding how transcription is regulated is to have full knowledge of the complete set of factors and events that take place throughout the various steps in the transcription cycle. While many key factors have been identified, we lack the links to gain a complete view in molecular terms of the steps required to achieve activation or repression of a target gene. A major gap in our understanding has been due to the absence of an isolation technique based on the genomic region of interest rather than the regulatory proteins themselves. By adapting a method recently developed by the Kingston laboratory to isolate specific regions of the genome along with their associated proteins, we will study in detail how specific genes are activated and repressed in response to environmental stress in the form of heat shock using the fruit fly Drosophila melanogaster. The robustly regulated heat shock response in Drosophila is ideally suited to analyze the controlled, rapid activation and repression of transcription and to uncover general mechanisms of transcriptional regulation that also occur in human cells. This study will allow for unbiased identification of new transcription factors, and permit the characterization of known factors according to their temporal roles. The elucidation of the heat shock pathway will provide valuable new information that will allow for the restructuring of current models of transcriptional regulation and will open the door for future functional studies. By characterizing in molecular detail the normal activation and repression of stress response genes, we can begin to understand how these processes are disrupted in cancers. ) PUBLIC HEALTH RELEVANCE: Precise control over gene expression is required to ensure that cells behave appropriately during normal processes and also respond to external stimuli quickly, and defects in gene expression can directly and indirectly lead to the development of cancer. Examples of abnormal function of gene regulatory factors have been found in almost all human cancers, so it is important to understand exactly how these factors work to turn on or off a particular gene at the appropriate time and place. The goal of this project is to provide an unbiased, unprecedented view of the molecular events leading to changes in gene expression at specific genes. A well-defined model system will be used to gain detailed information about the factors and steps required to respond appropriately to cellular stress, while also uncovering general mechanisms of gene regulation. This will lead to an overall better understanding of gene regulation under normal conditions, and importantly, what goes wrong in the development of disease.